Epstein-Barr virus (EBV) was discovered in 1964 in the neoplastic B cells of a patient with Burkitt's lymphoma. EBV thus became the first candidate for a human tumor virus. Early studies indicated the viral genome was present in two endemic tumors, Burkitt's lymphoma (equatorial Africa) and nasopharyngeal carcinoma (Southern China and coastal Asia) (Henle W. & Henle G. (1985) Epstein-Barr virus and human malignancies, Adv. Viral Oncol. 5, 201). By the late 1970s, it became evident that EBV plays a role in the development of B-cell lymphoproliferative disorders/lymphomas (BLPD) in T-cell immunocompromised patients, including solid organ and bone marrow transplant recipients, patients infected with HIV and children with congenital immunodeficiencies (Hanto D. W., Gajl-Peczalska K. J., Frizzera G., Arthur D. C., Balfour H. H., Jr., McClain K. (1983) EBV-induced polyclonal and monoclonal B-cell lymphoproliferative diseases occurring after renal transplantation. Clinical, pathologic, and virologic findings and implications for therapy, Ann Surg. 198, 356-69). In recent years, EBV has been identified in the hematopoietic tumor cells of approximately 50% of cases of Hodgkins disease, a large number of T-lymphomas and rare NK/monocytoid/dendritic cell malignancies, as well as in sporadic epithelial carcinomas (including in particular gastric carcinoma, and possibly aggressive breast carcinomas) (Kieff E. (2001) EBV and its Replication, In: Fields Virology, Phila., Pa.: Lippincott-Raven; p. 2511-2573; Pagano J. S. (1999) EBV: the first human tumor virus and its role in cancer, Proc. Assoc. Am. Physicians, 111, 573-580; Bonnet M., Guinebretiere J. M., Kremmer E., Grunewald V., Benhamou E., Contesso G. (1999) Detection of EBV in invasive breast cancers, J. Natl. Cancer Inst. 91, 1376-1381). An association with leiomyosarcomas in immunodeficient patents has also been found (Rogatsch H., Bonatti H., Menet A., Larcher C., Feichtinger H., Dirnhofer S. (2000) EBV-associated multicentric leiomyosarcoma in an adult patient after heart transplantation: case report and review of the literature. Am. J. Surg. Path. 24, 614-21). Several lines of evidence indicate that viral gene products contribute to multi-step tumorigenesis in these diverse neoplasms.
EBV is a member of the human herpesvirus family. Infection in childhood is usually asymptomatic; however, approximately 50% of individuals with delayed exposure develop a self-limited lymphoproliferative syndrome, acute infectious mononucleosis. Similar to other herpesviruses, EBV persists in a latent form for the life of the host. Serological surveys indicate that greater than 90% of the world population is infected with EBV (Henle W. & Henle G. (1979) Seroepidemiology of the virus, In: The Epstein-Barr Virus (Epstein M. A. & Achong B. G. eds), Springer Verlag, New York, pp. 61-73). The ubiquity of infection, coupled with increasing evidence for a broad role in virus-associated malignancies (in the immuno-compromised as well as the normal host) demonstrates a critical need to develop preventive and therapeutic strategies to limit EBV infection and the effects thereof.
Several approaches to prevent and treat the manifestations of EBV-associated malignancies are being investigated. These include generation of vaccines (for prevention, though one does not exist at this time), delivery of humoral and cell-based immune therapies, chemotherapy, gene therapy (for treatment) and antiviral drug therapy, based on decreasing EBV lytic replication in the hope that this will indirectly result in a decrease in the number of EBV-infected cells able to develop into tumors (for prophylaxis).
Treatment of EBV and EBV-Associated Diseases
Immunomodulatory agents such as α- and γ-interferons, WIG, retinoic acid, and others, either alone or in combination have been used to treat B-cell lymphoproliferative disease (BLPD), with variable success. However, responses have not been observed in other EBV-associated tumors (Shapiro R. S., Chauvenet A., McGuire W., Pearson A., Craft A. W., McGlave P., Filipovich A. (1988) Treatment of B-cell lymphoproliferative disorders with interferon alfa and intravenous gamma globulin, N. Engl. J. Med. 318, 1334; Pomponi F., Cariati R., Zancai P., De Paoli P., Rizzo, S., Tedeschi, R. M. (1996) Retinoids irreversibly inhibit in vitro growth of EBV-immortalized B lymphocytes, Blood, 88, 3147-3159).
The use of complement-fixing, anti-B cell monoclonal antibodies to treat EBV-infected B-lymphomas previously achieved limited success. A B-cell-directed Mab to CD20 (Rituximab) is now commercially available and has achieved the greatest success in treatment of BLPD/lymphoma to date. However, anaphylactic reactions have limited therapy in some cases and not all B-cell tumors bear CD20. Moreover, Rituximab confers no specificity for the virus-infected cell, causing long-term impairment of new humoral immune responses; Rituximab also has no efficacy for a spectrum of non-B-cell, EBV-associated diseases (Fischer A., Blanche S., Le Bidois J., Bordigoni P., Garnier J. L., Niaudet P. (1991) Anti-B-cell monoclonal antibodies in the treatment of severe B-cell lymphoproliferative syndrome following bone marrow and organ transplantation, N. Engl. J. Med. 324, 1451-1456; Milpied N., Vasseur B., Parquet N., Garnier J. L., Antoine C., Quartier P. (2000) Humanized anti-CD20 monoclonal antibody (Rituximab) in post transplant B-lymphoproliferative disorder: a retrospective analysis on 32 patients, Ann. Oncol. 11 (Suppl 1), 113-116).
Individual cell-based immune strategies are promising, but they incur a risk of graft-versus-host-disease and of transmission of pathogens during ex vivo propagation/preparation of cells, and will be expensive (Papadopoulos E. B., Ladanyi M., Emanuel D., Mackinnon S., Boulad F., Carabasi M. H. (1994) Infusions of donor leukocytes to treat EBV-associated lymphoproliferative disorders after allogeneic BMT, N. Engl. J. Med. 330, 1185-1191; Aguilar L. K., Rooney C. M., Heslop H. E. (1999) Lymphoproliferative disorders involving EBV after hemopoietic stem cell transplantation, Curr. Opin. Oncol. 11, 96-101).
Gene therapy strategies to introduce novel compounds that inhibit EBV oncoproteins or that inhibit cellular genes that are critical for virus-associated oncogenesis or that introduce cytotoxic gene products (such as the HSV1-TK gene into EBV-infected tumor cells followed by ganciclovir therapy) are under study. All are in early development and suffer from standard problems of delivery to the appropriate tumor site.
In the chemotherapy field, currently there are few or no clinically effective anti-EBV agents that are without undesirable side effects. Although several drug candidates have been shown to be effective against EBV replication in cell culture, their clinical application has been restricted by their lack of effect on the course of latency associated disease, because all of these antiviral agents target only EBV replication.
The greatest challenge in EBV therapy is the latent infection. There are no drugs, licensed or even experimental, regardless of mechanism of action, that have shown any specific effect on latent EBV or other gamma herpesvirus infection (Lin, J. C. (1999) Antiviral therapy for Epstein-Barr virus: the challenge ahead, Recent Res. Develop. Antimicrob. Agents and Chemother. 3, 191-223; Pagano J. S. (1995) Epstein-Barr virus: therapy of active and latent infection, in Antiviral Chemotherapy (eds. Jeffries & De Clercq), John Wiley & Sons, Chichester, pp. 155-195).
Several compounds have been shown to have activity against EBV replication in culture at concentrations non-toxic to cell growth. These include acyclovir (ACV), ganciclovir (DHPG), pencyclovir, D-FMAU and its analogs, 5-bromovinyl dUrd, phosphonoformate and phosphorothioate oligonucleotides. See Lin et al., Antimicrob. Agents Chemo. 32:265-267 (1988); Lin et al., Antimicrob. Agents Chemo., 32:1068-1072 (1988); Cheng et al., Proc. Natl., Acad. Sci. USA, 80:2767-2770 (1983); Datta et al., Proc. Natl., Acad. Sci. USA, 77:5163-5166 (1980); Datta et al., Virol., 114:52-59 (1981); Lin et al., Antimicrob. Agents & Chemo., 31:1431-1433 (1987); Olka & Calendar, Virol. 104:219-223 (1980); Lin et al., J. Virol., 50:50-55 (1984); Yao et al., Antimicrob. Agents & Chemo. 37:1420-1425 (1993) and Yao et al., Biochem. Pharm., 51:941-947 (1966).
U.S. Pat. Nos. 5,565,438, 5,567,688 and 5,587,362 (Chu et al.) disclose the use of 2′-fluoro-5-methyl-β-L-arabinofuranolyluridine (L-FMAU) for the treatment of hepatitis B and Epstein-Barr virus.
WO96/13512 (Genencor International, Inc. and Lipitek, Inc.) discloses the preparation of L-ribofuranosyl nucleosides as antitumor agents and virucides.
Tsai et al. in Biochem. Pharmacol. 1994, 48(7), 1477-81, disclose the effect of anti-HIV agents 2′-β-D-F-2′,3′-dideoxynucleoside analogs on the cellular content of mitochondrial DNA and lactate production.
WO 96/40164 and WO 95/07287 (Emory University, UAB Research Foundation, and the Centre National de la Recherche Scientifique) disclose several β-L-2′,3′-dideoxynucleosides for the treatment of hepatitis B virus and HIV, respectively.
Novirio Pharmaceuticals, Ltd. (WO 00/09531) disclose 2′-deoxy-β-L-erythropento-furanonucleosides (also referred to as β-L-dN or β-L-2′-dN), including β-L-deoxyribothymidine (β-L-dT) and β-L-deoxyribouridine (β-L-dU).
U.S. Pat. Nos. 5,792,773, 6,022,876 and 6,274,589 (Yale University and The University of Georgia Research Foundation, Inc.) disclose certain β-L dioxolanyl uracil-based nucleosides for the treatment of EBV. The compounds preferably have a 5-halosubstituted uracil base, and reportedly exhibit unexpectedly high activity against EBV, Varicella-Zoster virus (VZV) and Kaposi's Sarcoma virus (HV-8).
Gene Therapy
In studies of gene therapy for cancer, researchers are working to recruit the immune response to fight the disease or to make the cancer cells more sensitive to ablative treatment, such as chemotherapy. Some of the gene therapy techniques under study include:                Substitution of a “working” copy of a gene for an inactive or defective gene. For example, this technique could be used to restore the ability of a defective gene (such as mutant p53) to suppress or block the development of cancer cells.        Introduction of a “survival gene,” such as the multidrug resistance (MDR) gene into stem cells (cells in the bone marrow that produce blood cells). The MDR gene is used to make the stem cells more resistant to the side effects of the high doses of anticancer drugs.        Injection of cancer cells with a gene that makes them more susceptible to treatment with an anticancer drug. Scientists hope that treatment with the drug will kill only the cells that contain the drug-sensitive gene. This is known as suicide gene therapy.        
Suicide gene therapy is defined as the transduction of a gene that converts a prodrug into a toxic substance. Independently, the gene product and the prodrug are nontoxic. Two such systems have been widely investigated: the Escherichia coli cytosine deaminase (CD) gene plus 5-fluorocytosine (5-FC) and the herpes simplex virus thymidine kinase gene (HSV1-TK) plus ganciclovir (GCV).
The CD gene product converts 5-FC to the chemotherapeutic agent, 5-fluorouracil (5-FU) (Huber Be Austin, E A Good, S S, et al. (1993) In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res. 53:4619-4626) and has been studied primarily as a treatment for hepatic metastases of gastrointestinal tumors, for which 5-FU is commonly used. A significant bystander effect is active through production of locally high levels of freely diffusible 5-FU (Trinh Q T, Austin E A, Murray D M, et al., Enzyme/prodrug gene therapy: Comparison of cytosine deaminase/5-fluorcytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. Cancer Res 55:4808-4812, 1995). Systemic therapy with 5-FC results in growth suppression of CD-transduced tumors, whereas little growth inhibition is achieved in the same tumors with high doses of systemic 5-FU (Huber B E, Austin E A, Good S S et al. (1993) In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res. 53:4619-4626). No systemic growth suppression was seen in non-transduced tumors growing in the same animals, indicating the lack of serum 5-FU levels sufficient for antitumor activity. Interestingly, other investigators have noted that successful treatment of CD-transduced tumors with 5-FC can result in activity against challenge tumors (Mullen C A, Kilstrup M, Blaese R M (1992) Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc. Natl. Acad. Sci. USA 89:33-37). Depletion of CD8+ T-cells or granulocytes abrogates the effects of CD plus 5-FC (Consalvo M., Mullen C. A., Modesti A., Musiani P., Allione A., Cavallo F., Giovarelli M., Formi G. (1995) 5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory. J. Immunol. 154, 5302-5312), indicating that inadvertent stimulation of immunological activity in this system may further enhance the efficacy of this approach.
Strategies for treating liver metastases have focused on regional delivery of the CD gene into areas surrounding metastases (Ohwada A, Hirschowitz E A, Crystal R G (1996) Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver. Hum. Gene Ther. 7:1567-1576). Further refinements for systemic gene delivery are being explored through the use of tissue-specific promoters, such as carcinoembryonic antigen (CEA) or a fetoprotein genes, for targeting gene expression to liver tumor cells after hepatic-artery infusion of the CD vector (Richards C A, Austin E A, Huber B E (1995) Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy. Hum. Gene Ther. 6:881-893; Kanai F, Lan K H, Shiratori Y, et al. (1997) In vivo gene therapy for α-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. Cancer Res. 57:461-465).
The selective toxicity of ganciclovir (GCV) for cells expressing HSV-1-TK has been utilized similarly to promote tumor killing in a gene therapy model. In an original report (Culver K. W., Ram Z., Wallbridge S., Ishii H., Oldfield E. H., Blaese R. M. (1992) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors, Science, 256, 1550-1552), rapidly dividing murine glioma cells were infected in vivo with an amphotropic retrovirus vector containing HSV-TK. The animals were then treated with GCV. This resulted in the death of tumor cells expressing the viral TK, but spared adjacent normal cells that replicated too slowly for efficient retroviral infection. Because of the so-called bystander effects, this treatment is effective in destroying the tumor cells that contain as few as 10% TK-expressing cells. Adjacent cells that replicate rapidly also take up the cytotoxic phosphorylated nucleosides.
HSV1-TK phosphorylates GCV to GCV-monophosphate (GCV-MP) in a rate-limiting step, which can be further converted to a nucleotide analogue that inhibits DNA synthesis via cellular enzymes (Moolten F. L. (1986) Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res. 46, 5276-5281). This metabolic change causes a significant by-stander effect through several mechanisms: gap junctions transport non-diffusible phosphorylated GCV to non-transduced cells; non-transduced cells endocytose debris containing phosphorylated GCV from dying cells; and an induced immune response leads to tumor killing (Vile R. G., Nelson J. A., Castleden S., Chong H., Hart I. R. (1994) Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component. Cancer Res. 54, 6228-6234; Elshami A A, Saavedra A, Zhang H, et al. Gap junction plays a role in the “bystander effect” of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther 1996; 3:85-92; Hamel W, Magnelli L, Chiarugi V P, Israel M A. Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells. Cancer Res 1996 56:2697-2702; Mesnil M.; Piccoli C.; Tiraby G., Willecke K.; Yamasaki H. Bystander killing of cancer cells by herpes-simplex virus thymidine kinase gene is mediated by connexins. Proc. Natl. Acad. Sci. USA 93, 1831-1835; 1996). This type of gene therapy has been explored for a variety of cancers, including localized brain tumors (Culver K W, Ram Z, Walbridge S, Ishii H, Oldfield E H, Balese, R M 1992. In vivo gene transfer with retroviral vector producer cells for treatment of experimental brain tumors. Science 256:1550-1552; Barba D. Hardin J, Sadelain M, et al. Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci USA 1994; 91, 4348-52; Chen S, Shine H D, Goodman J C, Grossman R G, Woo S L C 1994. Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad Sci USA 91: 3054-3057) and mesotheliomas (Elshami A A, Saavedra A, Zhang H, et al. Gap junction plays a role in the “bystander effect” of the herpes simplex virus thymidine kinase/ganciclovir system in vitro. Gene Ther 1996; 3:85-92), liver metastases (Caruso M., Panis Y., Gagandeep S., Houssin D., Salzmann J. L, Klatzmann D. Regression of established macroscopic liver metastases after in-situ transduction of a suicide gene. Proc. Natl. Acad. Sci. USA 90, 7024-7028, (1993)), and peritoneal-based metastases (Tong X W, Block A, Chen S H, Woo S L C, Kieback D G. Adenovirus-mediated thymidine kinase gene transduction in human epithelial ovarian cancer cell lines followed by exposure to ganciclovir. Anticancer Res 1996 16, 1611-1617; Yee D, McGuire S E, Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo S L C. Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. Hum Gene Ther 1996 7, 1251-1257). There have been more than 35 clinical trials using this approach for human cancers worldwide. Although the growth-suppressive activities of HSV-TK plus GCV are significant, cure rates thus far are low, as in situ transduction (gene delivery) remains inadequate and the bystander effect is variable. Notably, both the CD and HSV-TK systems, and p. 53 gene therapy, additionally sensitize cancer cells to radiation, suggesting possible combination therapies to control advanced tumors (Kim J H, Kim S H, Kolozsvary A, Brown S L, Lim O B, Freytag S O. Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents. Int J Radiat Oncol Biol Phys 33: 861-868, 1995; Khil M. S.; Kim J. H., Mullein C. A., Kim S. H., Freytag S. O. Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene. Clinical Cancer Res. 2, 53-57 (1996)).
Use of HSV-TK plus GCV for the treatment of metastatic disease presents several problems. Treatment of tumors with HSV-TK suppresses growth of tumors derived from challenge injections of the parental cell line, indicating the induction of systemic anti-tumor activity in some models (Barba D. Hardin J, Sadelain M et al. Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors. Proc Natl Acad Sci USA 1994 91, 4348-52; Vile R. G., Nelson J. A., Castleden S., Chong H., Hart I. R. Systemic gene-therapy of murine melanoma using tissue-specific expression of the HSVtk gene involves an immune component. Cancer Res. 54, 6228-6234; 1994). Some evidence exists that this suppression is mediated by immune cells (Vile R. G., Nelson J. A., Castleden S., Chong H., Hart I. R. Systemic gene-therapy of murine melanoma using tissue-specific expression of the HSVtk gene involves an immune component. Cancer Res. 54, 6228-6234; 1994; Yamamoto S., Suzuki S., Hoshino A., Akimoto M., Shimada T. (1997) Herpes simplex virus thymidine kinase/ganciclovir-mediated killing of tumor cells induces tumor-specific cytotoxic T-cells in mice. Cancer Gene Ther. 4, 91-96), but the significance and generality of these observations are largely unknown. Furthermore, systemic delivery of HSV-tk to target metastatic lesions through intravenous (Vile R. G., Nelson J. A., Castleden S., Chong H., Hart I. R. Systemic gene-therapy of murine melanoma using tissue-specific expression of the HSVtk gene involves an immune component. Cancer Res. 54, 6228-6234; 1994) or peritoneal (Tong X W, Block A, Chen S H, Woo S L, Kieback D G. Adenovirus-mediated thymidine kinase gene transduction in human epithelial ovarian cancer cell lines followed by exposure to ganciclovir. Anticancer Res 1996 16: 1611-1617; Yee D, McGuire S E, Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo S L. Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. Hum Gene Ther 1996; 7, 1251-1257) routes may lead to significant liver injury (Yee D, McGuire S E, Brunner N, Kozelsky T W, Allred D C, Chen S H, Woo S L C. Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. Hum Gene Ther 1996; 7, 1251-1257; Brand K., Arnold W., Bartels T., Lieber A., Kay M. A., Strauss M., Dorken B. Liver-associated toxicity of the HSV-tk/GCV approach and adenoviral vectors. Cancer Gene Therapy 4, 9-16; 1997; Qian C, Idoate M, Bilbao R, Sangro B, Bruna O. Vazquez J et al. Gene transfer and therapy with adenoviral vector in rats with diethylnitrosamine-induced hepatocellular carcinoma. Hum Gene Ther 1997; 8, 349-358); tissue-specific vectors may be required for safe systemic delivery of this gene.
Epstein Barr Virus Thymidine Kinase (EBV-TK)
EBV-associated diseases are primarily manifest as virus-infected tumors in which the viral genome is present, however, infection is latent and few EBV genes are expressed. Increased levels of EBV lytic replication have been observed in the setting of acute infectious mononucleosis, a usually self-limited lymphoproliferative disorder and in oral hairy leukoplakia, a lytic disease of the oral cavity that occurs primarily in patients with AIDS. Increased levels of EBV lytic replication have been observed in the blood of immunocompromised patients and correlates with the subsequent development of lymphoproliferative disorders in these patients. There is no standard therapy for any of these conditions.
EBV encodes a thymidine kinase (TK), which is strictly a kinase with the capacity to phosphorylate thymidine and a range of thymidine analogs that is localized to the BamHI X fragment of the genome (Tung P. P. & Summers W. C. (1994) Substrate specificity of Epstein-Barr virus thymidine kinase. Antimicrob. Agents Chemother. 38, 2175-79; Gustafson E. A., Chillemi A. C., Sage D. R., Fingeroth J. D. (1998) The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow specificity compared to herpes simplex virus type 1 thymidine kinase. Antimicrob. Agents Chemother. 42, 2923-31). Although latently EBV-infected B-cells and EBV+ tumors do not routinely express EBV-TK, in vitro exposure of latently infected cells to the tumor promoter (12-O-tetradecanoylphorbol-13-acetate) PMA/TPA or to the polar organic compound sodium butyrate (NaB) results in modest induction of lytic replication (1%-40% of cells depending on the line) that is accompanied by EBV-TK expression (Stinchcobe T. & Clough W. (1985) EBV induces a unique pyrimidine 2′-deoxynucleoside kinase activity in superinfected and virus producer B cell lines. Biochemistry, 24, 2021-2033). In several EBV+ B-cell lines, the use of TPA and NaB together has been found to synergistically activate the lytic cycle (Anisimova E., Prachova K., Roubal J., Vonka V. (1984) Effects of n-butyrate and phorbol ester (TPA) on induction of EBV antigens and cell differentiation. Arch. Virol. 81, 223-237). Even when production of virus is minimal, EBV genes active during the lytic cycle are induced by drug treatment, suggesting that drug-induced gene expression is not identical to expression triggered in the course of a normal, productive infection. Although herpesvirus replication proceeds in a defined sequence, with synthesis of immediate early (IE) genes preceding early (E) and late (L) genes, artificial inducers of productive infection can alter the normal stoichiometry of viral gene expression. Induction is often abortive with IE, and E proteins synthesized in the absence of late proteins and virus assembly. In recent studies, NaB and arginine butyrate (ArgB) and related compounds have been administrated to healthy adults and children without major side-effects (Daniel P., Brazier M., Cerutti I., Pieri F., Tardivel I., Desmet G. (1989) Pharmacokinetic study of butyric acid administered in vivo as sodium and arginine butyrate salts. Clin. Chim. Acta 181, 255-263; Perrine S. P., Ginder G. D., Faller D. V., Dover G. H., Ikuta T., Witkowska H. E. (1993) A short-term trial of butyrate to stimulate fetal globin-gene expression in beta-globin disorders. N. Engl. J. Med. 328, 81-86). ArgB is FDA approved to induce fetal hemoglobin and abort hemolytic crisis in children with sickle cell anemia and β-thalassemia (Perrine S. P., Ginder G. D., Faller D. V., Dover G. H., Ikuta T., Witkowska, H. E. (1993) A short-term trial of butyrate to stimulate fetal-globin-gene expression in the beta-globin disorders. N Engl. J. Med. 328, 81-86). In addition, protein kinase C activators pharmacologically distinct from TPA, such as the bryostatins, which lack tumor-promoting activity, are currently in clinical trials for cancer patients (Prendiville J., Crowther D., Thatcher N., Woll P. J., Fox B. W., McGown A. (1993) A phase I study of intravenous bryostatin 1 in patients with advanced cancer. Br. J. Cancer 68, 418-424).
Related gammaherpesvirus HHV8 expresses a thymidine kinase with similar substrate specificity to Epstein Barr virus (Gustafson E A, Schinazi R F, Fingeroth J D (2000) Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir. J. Virol. 74, 684-692)). This virus is currently referred to in the literature as KHSV. See also Moore S M, Cannon J S, Tanhehco Y C, Hamzeh F M, Ambinder R F. (2001) Induction of Epstein-Barr virus kinases to sensitize tumor cells to nucleoside analogues. Antimicrob Agents Chemother, 45(7):2082-91; Ansari A, Emery V C. (1999) The U69 gene of human herpesvirus 6 encodes a protein kinase which can confer ganciclovir sensitivity to baculoviruses. J Virol, 73(4):3284-91; Cannon J S, Hamzeh F, Moore S, Nicholas J, Ambinder R F. (1999) Human herpesvirus 8-encoded thymidine kinase and phosphotransferase homologues confer sensitivity to ganciclovir. J Virol, 73(6):4786-93; and Emery V C, Griffiths P D. (2000) Prediction of cytomegalovirus load and resistance patterns after antiviral chemotherapy. Proc Natl Acad Sci 97(14): 8039-44.
Therefore, it is an object of the present invention to provide compounds, compositions and methods for the treatment or prophylaxis of a host, particularly a human patient or other host animal, infected with EBV (or the related gammaherpesvirus KHSV).
It is another object of the present invention to provide compounds, compositions and methods for the treatment or prophylaxis of a host, particularly a human patient or other host animal, suffering from a disease associated with the lytic replication of EBV (or the related gammaherpesvirus KHSV).
It is further another object of the present invention to provide compounds, compositions and methods for the treatment or prophylaxis of a host, particularly a human patient or other host animal, suffering from a disease associated with abnormally proliferating cells that are infected with EBV (or the related gammaherpesvirus KHSV).
It is another objective of the present invention to provide cell lines that can be used in gene therapy, in particular, cell lines transfected with EBV-TK, or the related gammaherpesvirus thymidine kinase (KHSV-TK).
It is a further objective of the present invention to provide compounds, compositions and methods for gene therapy of a host, particularly a human patient or other host animal exhibiting a cancer or infection related to Epstein Barr Virus.
Finally, it is an objective of the present invention to provide kits and assays to assess the effects of compounds and/or compositions in reducing abnormal cellular proliferation using transduced cell lines.